Cardiovascular disease, which includes coronary heart disease (CHD) and stroke, is the leading cause of death and disability in developed countries of the world. CVD is caused by clogging of arteries. Major accepted risk factors for CVD include age, gender, hypertension, smoking, diabetes, elevated blood low density lipoprotein cholesterol (LDL-C), and decreased blood high density lipoprotein cholesterol (HDL-C).
In order to assess CVD risk, an assay panel is utilized for testing an individual's risk factors. A typical risk assessment screening test includes measuring fasting levels of total cholesterol (TC), triglycerides (TG), HDL-C, calculated LDL-C, hemoglobin Alc, and glucose. However, such a risk assessment panel is limited to traditional risk factors and does not provide a complete assessment of CVD risk, or ways to optimize treatment.
There are other tests for general metabolic factors of kidney, liver, muscle and thyroid function that are often not performed. These include blood urea nitrogen or BUN, creatinine, BUN/creatinine ratio, albumin, globulin, albumin/globulin ratio, alkaline phosphatase, liver enzymes AST and ALT, creatine kinase (CK), thyroid stimulating hormone (TSH), glomerular filtration rate (GFR), calcium, total protein, total bilirubin, sodium, potassium, chloride, carbon dioxide and uric acid; however, these tests are not always consistently used, despite their importance in ruling out secondary causes of lipid abnormalities.
There are other tests for heart disease risk, such as testing for levels of non-HDL cholesterol, and very low density lipoprotein cholesterol (VLDL-C), and the total cholesterol/HDL cholesterol ratio; however, these test are not consistently used in CVD risk assessment.
In addition there are specialized tests for CVD risk which include testing for levels of direct LDL cholesterol, small dense LDL cholesterol (sdLDL-C), lipoprotein (a) or Lp(a), apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), fibrinogen, and homocysteine.
There are specialized testing for C-reactive protein (CRP with a highly sensitive test or hsCRP), lipoprotein associated phospholipase A2 (LpPLA2), N-terminal pro-brain natriuretic peptide (NT-proBNP), insulin, adiponectin, and glycosylated hemoglobin (HbA1C); however, they are not widely or consistently used in CVD risk assessment.
There are tests for plasma sterols, such as lathosterol, desmosterol, beta sitosterol, campesterol, and cholestanol; however, they are not widely or consistently used in CVD risk assessment.
In addition, genotyping of apolipoprotein E and Factor V Leiden provides valuable information about CVD and dementia risk, as well as risk of clot formation, but it is generally not utilized in CVD risk assessment.
Lipoproteins in serum or plasma are complexes of various lipids and proteins. The major lipoproteins based on ultracentrifugal separation are chylomicrons (CM), very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). These lipoproteins can also be fractionated by size, protein components, electrophoretic mobility, or any combination of these. If plasma is subjected to two separation methods, the major lipoprotein classes can further be separated into subclasses. These subclasses differ from each other in size, charge, chemical composition, and patho-physiologic importance.
The ultracentrifugal (UC) method separates lipoproteins based on their respective specific flotation rate (density) into HDL, LDL, VLDL, and CM, in decreasing density, respectively. However, the UC method is very labor intensive, requires a specialized laboratory, and is very expensive. Moreover, the high separating force (100,000 times of normal gravity) used in this method affects the integrity of the lipoprotein particles, therefore, the UC method produces a significant amount of artifacts (in vitro altered lipoproteins) that affect the result. In addition, even finer fractionation of the sample is necessary for relating fractions to diseases, but the additional fractionation step increases the production of artifacts. For these reasons, separation of plasma or serum lipoproteins by ultracentrifugation is neither feasible nor ideal for clinical diagnostic evaluation of plasma lipoproteins and cardiovascular disease (CVD) risk.
Size exclusion separation of HDL, as with fast low pressure liquid chromatography (FPLC), has no adequate resolution, needs a large quantity of plasma, and produces artifacts due to the excessive dilution of the plasma. Magnetic nuclear resonance (NMR) is another characterization technique that is widely used, primarily because of its speed; however, it is unclear how to interpret NMR signal data or what these data represent. One-dimensional non-denaturing gel electrophoresis is also used for characterizing lipoproteins, such as HDL and LDL. With this method, lipoproteins are separated only by size. With this method, separation between the preβ-mobility and α-mobility HDL particles is not achievable. Thus, this method does not allow for the accurate assessment of α-1 HDL, preβ-2, or preβ-1 HDL, all of which are important particles for CVD risk assessment.
HDL can protect against atherosclerosis in several ways. The most cited HDL function to protect against atherosclerosis is its participation in reverse cholesterol transport. During this process, HDL removes cholesterol from macrophages in the vessel wall, preventing the transformation of macrophages into foam cells, eventually preventing the build-up of fatty streaks and plaque in the vessel wall. The cholesterol that originated in the macrophages is then carried by HDL to the liver for ultimate excretion into the bile.
HDL is also an anti-oxidant and anti-inflammatory agent. Oxidative stress can cause inflammation in the vessel wall. The protein and lipid components of HDL can prevent LDL oxidation. This is a very important function because oxidized LDL is the major carrier of cholesterol to macrophages present in the vessel wall. Moreover, HDL has anti-inflammatory functions and participates in the immune response.
The different HDL particles have different pathophysiological relevance. The many different functions of HDL are not distributed evenly among the various HDL subclasses. The best illustration of this is that cells have several ways to remove excess cholesterol. The different HDL particles specifically interact with the different pathways depending on cell type, the expressed receptor protein type on the surface of the cell, and cellular cholesterol content. Also, the different HDL particles participate differently in the anti-oxidation and anti-inflammation processes based on the lipid and protein composition of the HDL particles.
Data from HDL- and CVD-related population-based studies reveal the following:                For every 1 mg increase in HDL cholesterol, there is a 2-3% reduction in CVD risk. [“High-density Lipoprotein Cholesterol and Cardiovascular Disease. Four Prospective American Studies.”, Gordon, D. J. et. al., Circulation, 1989, January; 79(1):8-15].        In the Framingham Offspring Study, in men free of CHD (n=1277) and men with CHD (n=169), for every 1 mg/dl increase in α-1 HDL there was a 26% decrease in risk of CHD (probability (“p”)<0.001), and HDL particles were superior to HDL-C values in predicting prevalence of CHD. [“High-density Lipoprotein Subpopulation Profile and Coronary Heart Disease Prevalence in Male Participants of the Framingham Offspring Study”, Asztalos, B. F., et. al., Arterioscler Thromb Vasc Biol. 2004, November; 24(11):2181-7. Epub 2004 Sep. 23].        Patients with CHD have lower HDL-C due to decreases in the large cholesterol-rich α-1 HDL (−39%) and increases in the small lipid-poor alpha α-3 HDL (+29%) and pre-β1 HDL (+16%) as compared to age- and gender-matched controls. [“Distribution of ApoA-I-containing HDL Subpopulations in Patients with Coronary Heart Disease”, Asztalos, B. F., et. al., Arterioscler Thromb Vasc Biol., 2000, December; 20(12):2670-6; and “High-density Lipoprotein Subpopulation Profile and Coronary Heart Disease Prevalence in Male participants of the Framingham Offspring Study”, as cited above].        In the Veterans Affairs HDL Intervention Study (VA-HIT), low levels of α-1 and α-2 HDL predicted recurrent CHD events (n=398) versus no recurrence (n=1097) in men selected for low HDL C (less than 40 mg/dl and presence of CHD. Low α-1 HDL was the most significant parameter predicting recurrence (p<0.001). [“Value of High-Density Lipoprotein (HDL) Subpopulations in Predicting Recurrent Cardiovascular Events in the Veterans Affairs HDL Intervention Trial”, Asztalos, B. F., et. al., Arterioscler Thromb Vasc Biol., 2005, October; 25(10):2185-2191].        
Two-dimensional gel electropheresis is a separation method, based on the combination of two principles of electrophoretic separation (in the first dimension, particles are separated by charge and in the second dimension by size) that is very useful for reproducibly separating HDL particles with high resolution. The method is quantitative by utilization of protein immuno-localization and image-analysis. As a result of employing this two-dimensional HDL separation method, different HDL particles have been associated with CVD risk in population-based cross-sectional studies. The two-dimensional gel electrophoresis technology is also useful in the diagnosis of the homozygous and heterozygous state for rare inherited HDL disorders, such as apoA-I/C-III/A-IV, apoA-I/C-III deficiency, isolated apoA-I deficiency, ABCA1 deficiency, LCAT deficiency, SRB1 deficiency, CETP deficiency, lipoprotein lipase deficiency, hepatic lipase deficiency, and endothelial lipase deficiency. Based on the scans generated using this technique, it has become possible to differentiate among the various HDL particles; this allows for very precise evaluation of the severity of CVD-risk. Patients who are carriers of one normal and one damaged gene (referred to as heterozygotes) of the above list also have reduced levels of HDL and premature CVD. Patients who are carriers of two damaged genes (referred to as homozygotes) of the above list generally have a very high risk for premature CHD. Patients with ABCA1 mutations have only small pre-β1 HDL particles with hypercatabolism of apoA-I and have premature CHD. Patients affected with apoA-I deficiency have no HDL and have strikingly premature CHD. Whereas, patients affected with LCAT deficiency have only preβ-1 and α-4 HDL particles, and are at moderate to high risk for CVD. Different mutations in the cholesterol ester transfer protein (CETP) can cause either increased or decreased CETP activity, resulting in different changes in HDL particles. High CETP activity results in low levels of large α-1 and high levels of the small preβ-1 HDL particles. High CETP activity is associated with significant increased risk for CVD. Low CETP activity, which may be due to mutations in the gene encoding CETP or to effects of various drugs, causes high levels of α-1 HDL and low levels of preβ-1 HDL. This HDL subpopulation profile (high α-1 and low preβ-1) is associated with protection against CVD. Various mutations in the genes encoding lipoprotein-, hepatic-, and secretory-phospholipases can also be detected and recognized by their specific HDL subpopulation profile using this method.
Most importantly, the HDL subpopulation profile can differentiate subjects with increased risk for CVD independent of the HDL-C level. This is very important, as some subjects or an entire ethnic group may have low HDL-C level without any history of elevated CVD risk due to the fact that these subjects have not only increased HDL catabolism, but also enhanced HDL function. These subjects have a normal HDL particle distribution. However, some subjects with high HDL-C may experience a CVD event due to low HDL catabolism or dysfunctional HDL as seen with a defective SRB1 function.
Similar to HDL, LDL can also be separated into particles having different sizes, most commonly separated into small dense (sd) LDL and large LDL particles. It is proven and widely accepted in the lipoprotein field that sdLDL-C is more atherogenic than large LDL-C. The most common method for separating LDL by size is electrophoresis. The quantification of different LDL fractions is based on lipid staining in the gel, followed by density scanning and integrating the area under the curve. The major disadvantages of this method are that it is labor and time consuming, and has poor resolution. A more recent method involves the use of a specific mixture of detergents for removing other lipoproteins, and then measuring cholesterol only in small dense LDL or sdLDL. This method is adaptable to high throughput automated analyzers, and it has been standardized.
Risk for CVD is significantly higher in subjects with impaired glucose homeostasis. Risk for CVD among type 2 diabetic patients is as high as the risk among subjects with elevated LDL-C level. There are several ways to determine glucose homeostasis including the measurement of fasting and post-prandial blood glucose levels, insulin levels, and hemoglobin-A1c (HbA1c) determinations. Currently, HbA1c is the most commonly used test to determine the severity of diabetes. The method needs red blood cells and fresh samples. Because the in-vivo half-life time of hemoglobin is about two to three months, measuring the amount of glucose attached to hemoglobin or HbA1c has been shown to be an excellent measure of long-term (8-12 weeks) blood glucose control. However, doctors who treat patients with CVD usually look for a shorter time period to determine whether the medications they prescribe affect diabetes. Moreover, there is not a wide range of values in the normal population. There is a way to measure shorter term changes in glucose homeostasis, namely by measuring glycated albumin (GA) as the percentile of plasma total albumin, which represents the glycation status over the past two to four weeks versus the three month period of HbA1c. This measurement is easy; utilizes plasma samples, and can be measured from stored (frozen) samples. Further, its value correlates well with HbA1c values, and due to the larger dynamic range of GA % measurement, subjects without known diabetes can be characterized more accurately with regard to their risk of developing diabetes and CVD. GA % measurement can also facilitate the diagnosis of pre-diabetes status.
Cardiovascular disease is considered both a lipid storage and an inflammatory disease. One of the inflammatory markers that have been shown to be an independent marker of CVD is C-reactive protein (CRP). CVD patients have increased CRP level. CRP has been a very well studied CVD-risk factor in the last couple of years. CRP is measured in plasma using a high sensitivity CRP assay kit. Recently, it has been found that CRP has several molecular forms (CRPmf) in human plasma. These forms differ in electrophoretic mobility and size, as assessed by polyacrylamide gel electrophoresis and immuno-localization under special conditions. The concentrations of the smallest molecular form (CRP mf4), or the ratio of this small CRP mf4 to the largest one (CRP mf1) is positively associated with fat cell mass (obesity) and with the presence of CVD.
Adiponectin is a protein hormone that modulates a number of metabolic processes, including glucose regulation and fatty acid catabolism. Adiponectin is exclusively secreted by adipose tissue into the bloodstream and is very abundant in plasma relative to many other hormones. Levels of the hormone are inversely correlated with body fat percentage in adults. The hormone plays a role in the suppression of the metabolic derangements that may result in type 2 diabetes, obesity, atherosclerosis and non-alcoholic fatty liver disease.
Despite the evident need for a better predictor of CVD, the market lacks a diagnostic solution consisting of a complete test panel for screening of an individual's CVD risk and a process for an accurate and individualized diagnosis and treatment plan derived from the results of the screening tests in order to optimize therapy and decrease CVD risk, especially in those patients who already have established CVD.